雷公藤内酯醇联合热疗对MKN28细胞增殖抑制作用的研究

Synergistic interaction of hyperthermia and TPL in gastric cancer cells

目的:研究雷公藤内酯醇(triptolid,TPL)与热疗联合应用对胃癌细胞MKN28增殖、细胞周期分布及磷酸化蛋白激酶B(phosphorylation of Akt,p-Akt)表达的影响。方法:将体外复苏和培养的胃癌细胞系MKN28分为四个组:对照组、TPL组、热疗组、TPL联合热疗组。其中TPL刺激浓度为20ng/ml,热疗采用43℃下加热150min。使用四甲基偶氮唑盐比色法(measurement of trititaed thymidine incorporation,MTT)检测细胞增殖并计算细胞增殖抑制率。流式细胞仪(flow cytometer,FCM)检测细胞周期分布的变化,使用蛋白质印迹法(Western blot)检测p-Akt水平。结果:热疗组,TPL组和TPL联合热疗组MKN28细胞增殖抑制率分别为21.2%,28.5%和54.5%,联合组显著高于TPL组和热疗组。FCM结果显示热疗组G0/G1期减少至(62.7±3.8)%,G2/M期增多至(18.1±3.2)%。TPL组G0/G1期增多至(80.62±4.6)%,G2/M期增多至(16.18±2.2)%。联合组G0/G1期(77.72±8.1)%,G2/M期增多至(19.62±2.4)%。热疗组和TPL组对细胞周期的影响不一样。与热疗组和TPL组相比,联合组G2/M期增多最明显。Western blot检测各组p-Akt蛋白表达均有降低,其中对照组(1.81±0.05),热疗组(1.28±0.01),TPL组(0.95±0.07),联合组(0.58±0.06),联合组下降最明显(P<0.05)。结论:TPL联合热疗对MKN28细胞增殖抑制作用更强,可能与二者对细胞周期的作用点不同,并能同时降低细胞内p-Akt的表达有关。

Objective：To explore the cytotoxic effects of TPL and hyperthermia in human gastric cancer cells in vitro.Methods：MKN28 cells were treated by four methods：control group,TPL group,hyperthermia group and TPL combined hyperthermia group.MTT assay was adopted to investigate the proliferation rates of cells.FCM was used to check the change of cell cycle in vitro.P-Akt of each group was checked by Western blot.Results：TPL combined hyperthermia group could significantly inhibit cell proliferation as compared with TPL group or hyperthermia group alone.The outcome of FCM indicated that TPL and hyperthermia affected cell-cycle distribution.The number of cells in G2/M phase had the greatest increase in TPL combined hyperthermia group.The level of p-Akt protein was lower in TPL group（0.95±0.07）,hyperthermia group（1.28±0.01） and combined group（0.58±0.06） when compared with those in the control group （1.81±0.05）,and the differences were significant（P〈0.05）.Conclusion：TPL with thermotherapy can enhance therapeutic effect on gastric tumor cells.It may be related with the distribution of cell cycle and the inhibition of expression of p-Akt.